The long-term goals of the research are to biochemically and genetically characterize the potential virulence factors used by intracellular mycobacteria to acquire iron from host cells. Mammals have an anti-infective system that attempts to withhold the biologically essential metal iron from invading microbes, but the "iron- withholding" defenses may be most effective against extracellular pathogens. Another possible defense system (called "iron-depletion" because it includes efflux of non-heme iron from infected host cells) may operate against pathogens living inside host cells. Successful pathogens must overcome these barriers by including effective iron acquisition among their virulence properties. Siderophore production is a common iron gathering tactic of microbes and in mycobacteria cultured extra- cellularly, a siderophore (mycobactin)-mediated iron uptake system is known. However, there is scant information on iron acquisition mechanisms used by mycobacteria present in macrophage phagosomes. Mycobactin may not be essential for intracellular iron uptake because pathogenic myco- bacterial strains lacking mycobactin production are known. The hypothesis is that the unidentified intracellular iron uptake mechanism(s) may be crucial for virulence of mycobacteria. The specific aims are: 1. to establish a mycobacteria/macrophage culture system that will accurately monitor the flow of iron to mycobacteria present within phagosomes, 2. to identify and characterize the mycobacterial factors and processes, as well as the genes encoding synthesis of these, that are essential for intracellular iron uptake.